Validation of novel CAR-T cell therapies against colorectal cancer

Abstract

Chimeric antigen receptor (CAR) T cell therapy has demonstrated a high degree of efficacy in patients with haematological malignancies such as acute lymphatic leukaemia. However, its efficacy in solid tumours remains to be demonstrated. There are a number of issues currently limiting therapeutic treatment of solid tumours with CAR-T cells, none more important than target antigen selection. This is a major challenge in solid tumours due to their heterogenous characteristics and potential off-cancer effects. ADAM10, A Disintegrin And Metalloproteinase 10, plays critical roles in a wide range of pathophysiological processes during development and inflammation. These proteolytically active multifunctional proteins act as sheddase by cleaving transmembrane-bound proteins such as growth factors, adhesion molecules and chemokine receptors, which can then contribute to migration and invasion of cancer cells. An active form of ADAM10 that has undergone a conformational change has been identified in several cancer types, particularly colorectal cancer. In this study, a novel CAR was generated based on a previously characterised conformation cancer-specific ADAM10 antibody. Another potential target for CAR-T therapy is leucine-rich repeat-containing G-protein coupled receptor (LGR5), which is a cancer stem cell marker and plays a significant role in the initiation and spreading of tumours. The research in this thesis examines the hypothesis that CAR-T cells directed against ADAM10 and LGR5 will kill colorectal cancer cells and inhibit growth of colorectal tumours in preclinical models. To test this hypothesis, both ADAM-10 and LGR5-targeting CAR-T cells were manufactured using T cells from the peripheral blood of the healthy donors. These CAR-T cells displayed the naïve and central memory phenotype that has been shown to differentiate and proliferate within tumour microenvironments, with little expression of exhaustion markers. These CAR-T cells were capable of killing several human colorectal tumour cell lines in vitro, assessed in a bioluminescence-based cytotoxicity assay. Furthermore, treatment of NSG mice bearing human LoVo colon cancer cell tumours with both ADAM-10 and LGR5-targeting CAR-T cells led to significant inhibition of tumour growth compared with either untransduced control T cells or PBS treatment. These data indicate that targeting ADAM-10 and LGR5 via CAR-T cell therapy is a promising novel avenue of treatment of colorectal cancer. The study also explored engineering the expression of chemokine receptors, with CXCR2 expression resulting in enhanced in vivo anti-tumour efficacy of LGR5-targeting CAR-T cells, but without impacting the efficacy of CXCR2 modified ADAM10-targeting CAR-T cells.